Patients with Alzheimer's disease initially show short term memory loss. As the disease progresses, the patients become completely demented. The brains of Alzheimer's patients contain numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates). Not surprisingly, such plaques and tangles are found primarily in those regions of the brain involved with memory and cognition.
The amyloid plaques are associated with dystrophic dendrites and axons, as well as activated nicroglia and reactive astrocytes. The major component of the plaques is a peptide referred to as A-beta (Selkoe, Science, 275:630-631 [1997]).
A-beta is ultimately generated by endoprotease cleavages of the polypeptide beta-amyloid precursor protein (beta-APP) Beta-APP is constitutively expressed in most cells of the body, but production of A-beta appears to be highest in brain cells. A-beta is typically secreted by the cells that produce it into the extracellular matrix where it makes its way into various bodily fluids. A schematic diagram of beta-APP processing is set forth in FIG. 12.
There are three major isoforms of beta-APP polypeptide, and they are referred to as beta-APP695, beta-APP751 and beta-APP770. The number on each isoform refers to the number of amino acids it contains.
Beta-APP is synthesized as a membrane protein and spans the Golgi membrane. Normal non-pathogenic processing of beta-APP is believed to occur via a putative enzyme referred to as “alpha-secretase”. Alpha-secretase cleaves between amino acids 687 and 688 of beta-APP770 (or between amino acids 612 and 613 of beta-APP695), thereby generating a 687 amino acid soluble form of beta-APP770 (or a 612 amino acid soluble form of beta-APP695) referred to as “alpha-APPs”. The remaining membrane-bound portion of beta-APP (amino acids 688-770 of the beta-APP770 isoform) is cleaved by the enzyme gamma-secretase. Gamma secretase cleaves between amino acids 711 and 712, and between amino acids 713 and 714, to generate the fragments 688-711, 688-713, and 712-770 and 714-770. The first two fragments are referred to as “p3” and are released from the membrane. Gamma secretase is also active on the two other isoforms of beta-APP.
In both non-diseased people and Alzheimer's disease patients, beta-APP is also processed via an alternative mechanism. Here, a heretofore uncharacterized enzyme referred to as “beta-secretase” cleaves full length beta-APP between amino acids 671 and 672 of beta-APP770, thereby generating a soluble fragment of amino acids 1-671, referred to as “beta-APPs” and a membrane bound form of amino acids 672-770. The membrane bound form is then acted on by gamma secretase which, as mentioned above, cleaves between amino acids 711 and 712, and between amino acids 713 and 714 to produce soluble fragments of amino acids 672-711 and 672-713; these fragments are referred to as “A-beta 40”, and “A-beta 42”, respectively. In addition, gamma secretase activity generates membrane bound fragments of amino acids 712-770 and 714-770. A-beta 40 and A-beta 42 are secreted from the cells and accumulate in the brains of Alzheimer's patients to form the characteristic brain tissue plaques.
Recent evidence suggests that beta secretase can also cleave beta-APP between amino acids 683 and 684 of the 770 isoform (Gouras et al., J. Neurochem., 71:1920-1925 [1998]) thereby ultimately generating a second form of A-beta spanning amino acids 684-711 and 684-713. The mechanisms that control this alternate cleavage are not known.
A few naturally occurring mutations of beta-APP have been identified. These include the so called “Swedish mutation” in which amino acids 670 and 671 of the 770 isoform are mutated, and the London mutation in which amino acid 717 of the 770 isoform is mutated. The Swedish mutation, which causes familial Alzheimer's disease, has been found to result in increased A-beta production in vivo (Citron et al., Nature, 360:672-674 [1992]; see also Citron et al., Neuron, 14:661-670 [1995]).
U.S. Pat. No. 5,744,346 purportedly describes a beta secretase molecule. No nucleic acid sequence or amino acid sequence for this molecule is presented.
European patent application EP O 855 444 A2 published 29 Jul. 1998 describes an aspartic proteinase that purportedly plays a role in Alzheimer's disease. This aspartic proteinase has sequence similarity at both the nucleic acid and amino acid levels to the beta-secretase of the present invention.
European patent application EP O 848 062 A2, published 17 Jun. 1998, describes an aspartic proteinase termed “ASP1” which has approximately 47 percent identity at the amino acid level to the prepro form of the beta-secretase of the present invention as determined by a computer comparison using the GAP alignment program.
Accordingly, it is an object of the invention to identify nucleic acid molecules encoding beta secretase polypeptides. Such molecules have use as probes for diagnosis of Alzheimer's disease, and for identification of compounds that modulate the activity of beta secretase.